Human Chemotactic Cytokine I Polypeptides

ABSTRACT

Human chemotactic cytokine I polypeptides and DNA (RNA) encoding such chemotactic cytokines and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such chemotactic cytokines for the treatment of leukemia, tumors, chronic infections, auto-immune disease, fibrotic disorders, wound healing and psoriasis. Antagonists against such chemotactic cytokines and their use as a therapeutic to treat rheumatoid arthritis, auto-immune and chronic and acute inflammatory and infective diseases, allergic reactions, prostaglandin-independent fever and bone marrow failure are also disclosed. Also disclosed are diagnostic assays for detecting diseases related to mutations in the nucleic acid sequences and altered concentrations of the polypeptides. Also disclosed are diagnostic assays for detecting mutations in the polynucleotides encoding the chemotactic cytokines and for detecting altered levels of the polypeptide in a host.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/994,821, filed Nov. 23, 2004, which is a continuation of U.S.application Ser. No. 09/227,854, filed Jan. 11, 1999, which is adivisional of U.S. application Ser. No. 08/761,289, filed Dec. 6, 1996,which claims benefit under 35 U.S.C. §119(e) of U.S. ProvisionalApplication No. 60/008,387, filed Dec. 8, 1995. This application is alsoa divisional of U.S. application Ser. No. 08/761,289, filed Dec. 6,1996, which claims benefit under 35 U.S.C. §119(e) of U.S. ProvisionalApplication No. 60/008,387, filed Dec. 8, 1995.

STATEMENT UNDER 37 C.F.R. § 1.77(b)(5)

This application refers to a “Sequence Listing” listed below, which isprovided in text format and entitled “PF210D1C2_SeqList.txt” (5,674bytes, created Apr. 19, 2007), which is hereby incorporated by referencein its entirety.

SUMMARY OF THE APPLICATION

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides. More particularly, the polypeptides of the presentinvention are human chemotactic cytokine I polypeptides. The inventionalso relates to inhibiting the action of such polypeptides.

BACKGROUND OF THE APPLICATION

The cytokine family of proteins exhibits a wide variety of functions. Ahallmark feature is their ability to elicit chemotactic migration ofdistinct cell types, including polymorphonuclear cells and macrophages.Many cytokines have proinflammatory activity and are involved inmultiple steps during inflammatory reactions. In addition to theirinvolvement in inflammation, cytokines have been shown to exhibit otheractivities. For example, interleukin-8 (IL-8) promotes proliferation ofkeratinocytes.

In light of the diverse biological activities, it is not surprising thatcytokines have been implicated in a number of physiological and diseaseconditions, including lymphocyte trafficking, wound healing,hematopoietic regulation and immunological disorders such as allergy,asthma and arthritis.

The S100 family of calcium binding proteins has chemotactic activity forpolymorphonuclear cells, mononuclear cells and neutrophils and arecalcium binding proteins. The S100 protein has been recently identifiedin cells of myeloid origin and consists of macrophage inhibitoryfactor-related protein (MRP-8), MRP-14, chemotactic protein 10 (CP-10)and calgranulin C.

MRP-8 and MRP-14 belong to the S100 family of proteins, which includescalbindin, and calcyclin (Kligman, D. and Hilt, D. C., Trends Biochem.Sci., 13:437-443 (1998)). This group of protein ranges in molecular sizefrom 10 to 14 kd and are also expressed in a cell lineage-specificmanner. Alignment of individual sequences shows that there is overallconservation of structure within the family, a notable feature being thetwo calcium binding sites, which are the “EF hand” type. Sequences atboth the NH₂— and COOH— terminal ends of MRP-8 and MRP-14 are relativelyhydrophobic. An attractive hypothesis is that these regions of themolecule are buried until calcium binding brings about theconformational changes that cause their exposure, making thempotentially available for interactions with other effector molecules.Because of the extended sequence of its COOH— terminal “tail” MRP-14 isthe largest member of the S100 family. (Hessian, P., et al., J. Leuk.Bio., 53:197-204 (1993).

Each gene in the S100 family is composed of three exons with one introninterrupting the protein-coding sequence between the two EF hands. TheMRP-8 and MRP-14 genes are both localized to chromosome 1Q12-Q21 with anundefined distance between them (Dorin, J. R., et al., Nature,326:614-617 (1987) and Lagasse, E. and Clerc, R. G., Mol. Cell. Biol.,8:2402, 2410 (1988)). Two other S100 family members 1882 (CAPL) andcalcyclin/2A9 (CACY) also map to chromosome 1Q12-Q21 (Dorian, J. R., etal., Genomics, 8:420-426 (1990). It is probably that co-segregation ofthese five genes on chromosome 1 may represent an S100 family locus.However, this does not apply to all S100 homologs. MRP-8 and MRP-14 arerestricted to cells of the monocytes/macrophage lineage, neutrophils,and under certain circumstance keratinocytes, suggesting that itsexpression is tightly regulated during differentiation (Hogg, N., etal., Eur. J. Immunol., 19:1053-1061 (1989)). Thus, monocytes andneutrophils in the circulation express MRP-8 and MRP-14, in contrast toother related cells such as lymphocytes, platelets, basophils andeocynophils which do not (Id.).

Resident tissue macrophages do not express MRP-8 and MRP-14, implyingthat differentiation of monocytes to macrophages is normally associatedwith loss of this molecule (Id.). Furthermore, immunohistochemical datashow that at inflammatory sites MRP-8 and MRP-14 positive cells areassociated with vessels but that the majority of monocytes alreadywithin the tissues at these sites have lost MRP-8 and MRP-14 expression(Id.). In keeping with these observations, tissue culture-maturedmonocytes down regulate this molecule (Zwadlo, G., et al., Clin. Exp.Immunol., 72:510-515 (1988)).

At sites of chronic inflammation in patients with diseases such asrheumatoid arthritis, sarcoidosis, tuberculosis or onchocerciasismacrophages express both MRP-8 and MRP-14 (Palmer, D. G., et al., Clin.Immunol. Immunopathol., 45:17-28 (1987)). In contrast, macrophages inacutely inflamed tissues may express only MRP-14 (Delabie, J., et al.,Clin. Exp. Immunol., 81:123-126 (1990)). The expression of MRP-8 andMRP-14 by macrophages could be flecked exposure to tissue stimuli thateither maintain expression or induce re-expression of the molecule(Palmer, D. G., et al., Clin. Immunol. Immunopathol., 45:17-28 (1987)).

In common with other members of the S100 family, MRP-8 and MRP-14 arefound predominately in a cytosolic location in both monocytes andneutrophils (Dale, I., et al., Eur. J. Biochem., 134:1-6 (1983)). It isalso possible that MRP-8 and MRP-14 can be expressed on the cellsurface, although the majority of antibodies specific for these proteinsdo not react with circulating monocytes or neutrophils. There is alsoevidence that MRP-8 and MRP-14 exist extracellularly, however, neitherprotein has the signal peptide sequence for membrane translocation.Thus, MRP-8 and MRP-14 fall into the category of proteins, includinginterleukin-1 and basic fibroblast growth factor, that clearly haveextracellular functions but about which little is known of theircellular release. Finally, MRP-8 and MRP-14 are found in the serum ofpatients with cystic fibrosis and other chronic inflammatory states suchas rheumatoid arthritis and sarcoidosis (Bullock, S., et al., Clin.Genet., 21:336-341 (1982)).

CP-10 is one of the most potent chemotactic proteins of the S100 family.An extracellular function of the murine CP-10 includes a potentchemotactic agent involved in phagocyte recruitment during inflammatoryreactions (Lackman, M., et al., J. Biol. Chem., 267:7499 (1992)). CP-10has an apparent molecular weight of 10.3 kd and a complete sequence of88 amino acids.

S100 proteins are characterized by two calcium binding regions, whichare strongly conserved and are separated by an 8 to 12 amino acid hingeregion (Kligman, D., Trends Biochem. Sci., 13:437 (1988)). Although thehinge region length is conserved, the amino acid sequences are widelydivergent. This divergence led to the hypothesis that the hinge regionmay concur functional specificity by interaction with the factorproteins (Id.).

In accordance with one aspect of the present invention, there areprovided novel polypeptides as well as biologically active anddiagnostically or therapeutically useful fragments, analogs andderivatives thereof.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules encoding such polypeptides,including mRNAs, cDNAs, genomic DNA as well as biologically active anddiagnostically or therapeutically useful fragments, analogs andderivatives thereof.

In accordance with another aspect of the present invention there isprovided an isolated nucleic acid molecule encoding a mature polypeptideexpressed by the DNA contained in ATCC™ Deposit No. 97304.

In accordance with another aspect of the present invention there areprovided nucleic acid probes comprising nucleic acid molecules ofsufficient length to specifically hybridize to sequences of the presentinvention.

In accordance with yet a further aspect of the present invention, thereis provided a process for producing such polypeptides by recombinanttechniques which comprises culturing recombinant prokaryotic and/oreukaryotic host cells, containing a nucleic acid sequence of the presentinvention, under conditions promoting expression of said protein andsubsequent recovery of said protein.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides for therapeutic purposes, forexample, to treat tumors, chronic infections, leukemia, T-cell mediatedauto-immune diseases, parasitic infections, psoriasis, asthma, allergy,to regulate hematopoiesis, to stimulate growth factor activity, toinhibit angiogenesis and to promote wound healing, to treat inflammatorydisorders, to control cellular immune reactions, to treat malignantdiseases, to inhibit casein kinase activity and to treatartherosclerosis.

In accordance with yet a further aspect of the present invention, thereare provided antibodies against such polypeptides and a method ofemploying such antibodies to detect diseases related to anoverexpression of the polypeptide of the present invention.

In accordance with yet another aspect of the present invention, thereare provided antagonists to such polypeptides, which may be used toinhibit the action of such polypeptides, for example, in the treatmentof certain auto-immune diseases, atherosclerosis, chronic inflammatoryand infectious diseases, histamine and IgE-mediated allergic reactions,prostaglandin-independent fever, bone marrow failure, cancers,silicosis, sarcoidosis, rheumatoid arthritis, shock, hyper-eosinophilicsyndrome and fibrosis in the asthmatic lung, cystic fibrosis, malignantdiseases, psoriasis, diapedesis and urinary and kidney stones.

In accordance with another aspect of the present invention there isprovided a method of diagnosing a disease or a susceptibility to adisease related to a mutation in the nucleic acid sequences and theprotein encoded by such nucleic acid sequences.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA and manufacture of DNAvectors.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIG. 1 displays the cDNA sequence and corresponding deduced amino acidsequence of the chemotactic cytokine I polypeptide of the presentinvention. The standard one-letter abbreviations for amino acids areused. Sequencing was performed using a 373 Automated DNA sequencer(Applied Biosystems, Inc.).

FIG. 2 is an illustration of amino acid sequence homology between thepolypeptide of the present invention and pig calgranulin C protein (SEQID NO:9).

FIG. 3 depicts the purification of the protein of the invention on agel.

In accordance with an aspect of the present invention, there areprovided isolated nucleic acids (polynucleotides) which encode for themature polypeptides having the deduced amino acid sequences of FIG. 1(SEQ ID No. 2).

Polynucleotides encoding the polypeptide of the present invention havebeen isolated from a human adult liver cDNA library. The polypeptidecontains an open reading frame encoding a protein of 92 amino acids. Theprotein exhibits the highest degree of homology at the amino acid levelto human MRP-14 with 46.739% identity and 67.391% similarity. Theprotein exhibits the highest degree of homology at the nucleotide levelto SP100 protein with 56% identity and 56% similarity.

As seen in FIG. 2 the polypeptide of the present invention retains thecalcium binding motifs present in all S100 protein members. Forinstance, amino acids, leucine and glutamine in amino acid position 4and 5, the leucine at position 8, and the leucine and lysine at position28 and 30 are important for calcium binding activity and are shown to bepreserved in the polypeptide of the present invention.

In accordance with another aspect of the present invention there areprovided isolated polynucleotides encoding a mature polypeptideexpressed by the DNA contained in ATCC™ Deposit No. 97304, depositedwith the American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 20110-2209, USA, on Sep. 25, 1995. The deposited materialis a pBluescript SK (−) plasmid (Stratagene, LaJolla, Calif.) whichcontains the full-length chemotactic cytokine I cDNA cloned into theEcoRI, XhoI site.

The deposit(s) have been made under the terms of the Budapest Treaty onthe International Recognition of the Deposit of Micro-organisms forpurposes of Patent Procedure. The strain will be irrevocably and withoutrestriction or condition released to the public upon the issuance of apatent. These deposits are provided merely as convenience to those ofskill in the art and are not an admission that a deposit is requiredunder 35 U.S.C. §112. The sequence of the polynucleotides contained inthe deposited materials, as well as the amino acid sequence of thepolypeptides encoded thereby, are incorporated herein by reference andare controlling in the event of any conflict with any description ofsequences herein. A license may be required to make, use or sell thedeposited materials, and no such license is hereby granted.

The polynucleotides of the present invention may be in the form of RNAor in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded, and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. The coding sequence which encodes the mature polypeptides may beidentical to the coding sequences shown in FIG. 1 (SEQ ID NO:1) or maybe a different coding sequence which coding sequence, as a result of theredundancy or degeneracy of the genetic code, encodes the same maturepolypeptides as the DNA of FIG. 1 (SEQ ID NO:1).

The polynucleotides which encode for the mature polypeptides of FIG. 1(SEQ ID NO:2) may include: only the coding sequence for the maturepolypeptide; the coding sequence for the mature polypeptide andadditional coding sequence such as a leader or secretory sequence or aproprotein sequence; the coding sequence for the mature polypeptide (andoptionally additional coding sequence) and non-coding sequence, such asintrons or non-coding sequence 5′ and/or 3′ of the coding sequence forthe mature polypeptides.

Thus, the term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes coding sequence for the polypeptide andmay also include additional coding and/or non-coding sequence such asintrons.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequencesof FIG. 1 (SEQ ID NO:2). The variant of the polynucleotides may be anaturally occurring allelic variant of the polynucleotides or anon-naturally occurring variant of the polynucleotides.

Further particularly preferred in this regard are polynucleotidesencoding human chemotactic cytokine I variants, analogs, derivatives andfragments, and variants, analogs and derivatives of the fragments, whichhave the amino acid sequence of the polypeptide of FIG. 1 or of thedeposit in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or noamino acid residues are substituted, deleted or added, in anycombination. Especially preferred among these are silent substitutions,additions and deletions, which do not alter the properties andactivities of the human chemotactic cytokine I. Also especiallypreferred in this regard are conservative substitutions. Most highlypreferred are polypeptides having the amino acid sequence of FIG. 1 orof the deposit; without substitutions.

Thus, the present invention includes polynucleotides encoding the samemature polypeptides as shown in FIG. 1 (SEQ ID NO:2) as well as variantsof such polynucleotides which variants encode for a fragment, derivativeor analog of the polypeptides of FIG. 1 (SEQ ID NO:2). Such nucleotidevariants include deletion variants, substitution variants and additionor insertion variants.

As hereinabove indicated, the polynucleotides may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequencesshown in FIG. 1 (SEQ ID NO:1). As known in the art, an allelic variantis an alternate form of a polynucleotide sequence which may have asubstitution, deletion or addition of one or more nucleotides, whichdoes not substantially alter the function of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the codingsequence for the mature polypeptides may be fused in the same readingframe to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5′ amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains.

Thus, for example, the polynucleotides of the present invention mayencode for a mature protein, or for a protein having a prosequence orfor a protein having both a prosequence and a presequence (leadersequence).

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptides of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptides fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

Fragments of the full length gene of the present invention may be usedas a hybridization probe for a cDNA library to isolate the full lengthcDNA and to isolate other cDNAs which have a high sequence similarity tothe gene or similar biological activity. Probes of this type preferablyhave at least 30 bases and may contain, for example, 50 or more bases.The probe may also be used to identify a cDNA clone corresponding to afull length transcript and a genomic clone or clones that contain thecomplete gene including regulatory and promotor regions, exons, andintrons. An example of a screen comprises isolating the coding region ofthe gene by using the known DNA sequence to synthesize anoligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 70%,preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term“stringent conditions” means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNA of FIG. 1 (SEQ ID NO:2).

Alternatively, the polynucleotide may have at least 20 bases, preferablyat least 30 bases, and more preferably at least 50 bases which hybridizeto a polynucleotide of the present invention and which has an identitythereto, as hereinabove described, and which may or may not retainactivity. For example, such polynucleotides may be employed as probesfor the polynucleotide of SEQ ID NO:1, for example, for recovery of thepolynucleotide or as a diagnostic probe or as a PCR primer.

Thus, the present invention is directed to polynucleotides having atleast a 70% identity, preferably at least 90% and more preferably atleast a 95% identity to a polynucleotide which encodes the polypeptideof SEQ ID NO:2 and polynucleotides complementary thereto as well asportions thereof, which portions have at least 30 consecutive bases andpreferably at least 50 consecutive bases and to polypeptides encoded bysuch polynucleotides.

The present invention further relates to polypeptides which have thededuced amino acid sequences of FIG. 1 (SEQ ID NO:2), as well asfragments, analogs and derivatives of such polypeptides.

The terms “fragment,” “derivative” and “analog” when referring to thepolypeptides of FIG. 1 (SEQ ID NO:2), means polypeptides which retainessentially the same biological function or activity as suchpolypeptides. Thus, an analog includes a proprotein which can beactivated by cleavage of the proprotein portion to produce an activemature polypeptide.

Among the particularly preferred embodiments of the invention in thisregard are polypeptides having the amino acid sequence of FIG. 1,variants, analogs, derivatives and fragments thereof, and variants,analogs and derivatives of the fragments. Alternatively, particularlypreferred embodiments of the invention in this regard are polypeptideshaving the amino acid sequence of the human chemotactic cytokine I ofthe cDNA in the deposited clone, variants, analogs, derivatives andfragments thereof, and variants, analogs and derivatives of thefragments.

Further particularly preferred in this regard are variants, analogs,derivatives and fragments, and variants, analogs and derivatives of thefragments, having the amino acid sequence of the polypeptide of FIG. 1or of the cDNA in the deposited clone, in which several, a few, 5 to 10,1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deletedor added, in any combination. Especially preferred among these aresilent substitutions, additions and deletions, which do not alter theproperties and activities of human chemotactic cytokine I. Alsoespecially preferred in this regard are conservative substitutions. Mosthighly preferred are polypeptides having the amino acid sequence of FIG.1 or the deposited clone without substitutions.

The polypeptides of the present invention may be recombinantpolypeptides, natural polypeptides or synthetic polypeptides, preferablyrecombinant polypeptides.

The fragment, derivative or analog of the polypeptides of FIG. 1 (SEQ IDNO:2) may be (i) one in which one or more of the amino acid residues aresubstituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or (iv) one in whichthe additional amino acids are fused to the mature polypeptide, such asa leader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polypeptides of the present invention include the polypeptide of SEQID NO:2 (in particular the mature polypeptide) as well as polypeptideswhich have at least 70% similarity (preferably at least 70% identity) tothe polypeptide of SEQ ID NO:2, and which have at least 90% similarity(more preferably at least 90% identity) to the polypeptide of SEQ IDNO:2 and still more preferably at least 95% similarity (still morepreferably at least 95% identity) to the polypeptide of SEQ ID NO:2 andalso include portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the genes. The culture conditions, such astemperature, pH and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila S2 andSpodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma;adenoviruses; plant cells, etc. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, pD10, phagescript, psiX174, pBluescript SK, pBSKS, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); pTRC99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene products encoded by the recombinant sequences.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), thedisclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples include the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC™ 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa, 293 and BHKcell lines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

The polypeptides can be recovered and purified from recombinant cellcultures by methods including ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsand diagnostics to human disease.

The human chemotactic cytokine I polypeptides may be employed to inhibitbone marrow stem cell colony formation as adjunct protective treatmentduring cancer chemotherapy and for leukemia.

The human chemotactic cytokine I polypeptides may also be employed toinhibit epidermal keratinocyte proliferation for treatment of psoriasis,which is characterized by keratinocyte hyper-proliferation.

The human chemotactic cytokine I polypeptides may also be employed totreat solid tumors by stimulating the invasion and activation of hostdefense cells, e.g., cytotoxic T cells and macrophages and by inhibitingthe angiogenesis of tumors. They may also be employed to enhance hostdefenses against resistant chronic and acute infections, for example,mycobacterial infections via the attraction and activation ofmicrobicidal leukocytes.

The human chemotactic cytokine I polypeptides may also be employed toinhibit T cell proliferation by the inhibition of IL-2 biosynthesis forthe treatment of T-cell mediated auto-immune diseases and lymphocyticleukemias.

The chemotactic cytokine I polypeptides may also be employed tostimulate wound healing, both via the recruitment of debris clearing andconnective tissue promoting inflammatory cells and also via its controlof excessive TGF β-mediated fibrosis. In this same manner, 1 may also beemployed to treat other fibrotic disorders, including liver cirrhosis,osteoarthritis and pulmonary fibrosis.

The human chemotactic cytokine I polypeptides of the present inventionmay also be employed as cytostatic agents for antibacterial andantimicrobial functions.

They may also be employed to regulate hematopoiesis, by regulating theactivation and differentiation of various hematopoietic progenitorcells, for example, to release mature leukocytes from the bone marrowfollowing chemotherapy.

The polynucleotides and polypeptides encoded by such polynucleotides mayalso be utilized for in vitro purposes related to scientific research,synthesis of DNA and manufacture of DNA vectors and for designingtherapeutics and diagnostics for the treatment of human disease.

The polypeptides of the present invention and fragments and analogs andderivatives thereof may be identified by assays which detect chemotacticactivity. One example of such an assay comprises testing suchpolypeptide for chemotactic activities toward murine polymorphonuclearcells or macrophages, human PMN (isolated on mono-poly-resolving medium;FLOW, McLean, V. A.). Conditioned medium (diluted and fully supplementedDulbecco's Modified Eagles medium) and cell lysates (diluted andsupplemented Dulbecco's Modified Eagles medium containing 0.1% BSAinstead of 10% FCS) from transiently transfected CV-1 cells are testedfor chemotactic activities toward murine PMNs. Endotoxin content ofmedia and all solutions to be tested are measured using a chromogeniclimulus amoebocyte lysate assay (Cape Cod Associates, Woods Hole,Mass.), which was sensitive to 5 pg endotoxin/ml. Chemotactic activityis defined as the mean number of cells migrating through the pores ofthe membrane in 3 to 5 standard fields and quantitated by image analysis(Wild-Leitz, Rockly, N.J.) using planimetry measurements (magnificationtimes 100) or by counting normally. Endotoxin-activated mouse serum (5%)or FMLP (10⁻⁷M) are used as positive controls.

This invention is also related to the use of the chemotactic cytokine Ipolypeptide gene as part of a diagnostic assay for detecting diseases-or susceptibility to diseases related to the presence of mutations inthe chemotactic cytokine I polypeptide nucleic acid sequences. Suchdiseases are related to under-expression of the human chemokinepolypeptides, for example, tumors and cancers.

Individuals carrying mutations in the chemotactic cytokine I polypeptidegene may be detected at the DNA level by a variety of techniques.Nucleic acids for diagnosis may be obtained from a patient's cells, suchas from blood, urine, saliva, tissue biopsy and autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986))prior to analysis. RNA or cDNA may also be used for the same purpose. Asan example, PCR primers complementary to the nucleic acid encodingchemotactic cytokine I polypeptide can be used to identify and analyzechemotactic cytokine I polypeptide mutations. For example, deletions andinsertions can be detected by a change in size of the amplified productin comparison to the normal genotype. Point mutations can be identifiedby hybridizing amplified DNA to radiolabeled chemotactic cytokine Ipolypeptide RNA or alternatively, radiolabeled chemotactic cytokine Ipolypeptide antisense DNA sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

The present invention also relates to a diagnostic assay for detectingaltered levels of chemotactic cytokine I polypeptide in various tissuessince an over-expression of the proteins compared to normal controltissue samples may detect the presence of a disease or susceptibility toa disease, for example, cystic fibrosis or malignancies such as cancersand tumors. Assays used to detect levels of chemotactic cytokine Ipolypeptide in a sample derived from a host are well-known to those ofskill in the art and include radioimmunoassays, competitive-bindingassays, Western Blot analysis, ELISA assays and “sandwich” assay. AnELISA assay (Coligan, et al., Current Protocols in immunology, 1(2),Chapter 6, (1991)) initially comprises preparing an antibody specific tothe chemotactic cytokine I polypeptide antigen, preferably a monoclonalantibody. In addition a reporter antibody is prepared against themonoclonal antibody. To the reporter antibody is attached a detectablereagent such as radioactivity, fluorescence or, in this example, ahorseradish peroxidase enzyme. A sample is removed from a host andincubated on a solid support, e.g. a polystyrene dish, that binds theproteins in the sample. Any free protein binding sites on the dish arethen covered by incubating with a non-specific protein like BSA. Next,the monoclonal antibody is incubated in the dish during which time themonoclonal antibodies attach to any chemotactic cytokine I polypeptideattached to the polystyrene dish. All unbound monoclonal antibody iswashed out with buffer. The reporter antibody linked to horseradishperoxidase is now placed in the dish resulting in binding of thereporter antibody to any monoclonal antibody bound to chemotacticcytokine I polypeptide. Unattached reporter antibody is then washed out.Peroxidase substrates are then added to the dish and the amount of colordeveloped in a given time period is a measurement of the amount ofchemotactic cytokine I polypeptide present in a given volume of patientsample when compared against a standard curve.

A competition assay may be employed wherein antibodies specific tochemotactic cytokine I polypeptide are attached to a solid support andlabeled chemotactic cytokine I polypeptide and a sample derived from thehost are passed over the solid support and the amount of label detected,for example by liquid scintillation chromatography, can be correlated toa quantity of chemotactic cytokine I polypeptide in the sample.

A “sandwich” assay is similar to an ELISA assay. In a “sandwich” assaychemotactic cytokine I polypeptide is passed over a solid support andbinds to antibody attached to a solid support. A second antibody is thenbound to the chemotactic cytokine I polypeptide. A third antibody whichis labeled and specific to the second antibody is then passed over thesolid support and binds to the second antibody and an amount can then bequantified.

This invention provides a method for identification of the receptors forthe human chemotactic cytokine I polypeptides. The gene encoding thereceptor can be identified by numerous methods known to those of skillin the art, for example, ligand panning and FACS sorting (Coligan, etal., Current Protocols in Immun., 1(2), Chapter 5, (1991)). Preferably,expression cloning is employed wherein polyadenylated RNA is preparedfrom a cell responsive to the polypeptides, and a cDNA library createdfrom this RNA is divided into pools and used to transfect COS cells orother cells that are not responsive to the polypeptides. Transfectedcells which are grown on glass slides are exposed to the labeledpolypeptides. The polypeptides can be labeled by a variety of meansincluding iodination or inclusion of a recognition site for asite-specific protein kinase. Following fixation and incubation, theslides are subjected to autoradiographic analysis. Positive pools areidentified and sub-pools are prepared and retransfected using aniterative sub-pooling and rescreening process, eventually yielding asingle clones that encodes the putative receptor.

As an alternative approach for receptor identification, the labeledpolypeptides can be photoaffinity linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE analysis and exposed to X-ray film. The labeledcomplex containing the receptors of the polypeptides can be excised,resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

This invention provides a method of screening compounds to identifyagonists and antagonists to the human chemotactic cytokine Ipolypeptides of the present invention. An agonist is a compound whichhas similar biological functions of the polypeptides, while antagonistsblock such functions. Antagonists and agonists may be identified by thechemotaxis assay described above.

Examples of potential chemotactic cytokine I polypeptide antagonistsinclude antibodies, or in some cases, oligonucleotides, which bind tothe polypeptides. Another example of a potential antagonist is anegative dominant mutant of the polypeptides. Negative dominant mutantsare polypeptides which bind to the receptor of the wild-typepolypeptide, but fail to retain biological activity.

Antisense constructs prepared using antisense technology are alsopotential antagonists. Antisense technology can be used to control geneexpression through triple-helix formation or antisense DNA or RNA, bothof which methods are based on binding of a polynucleotide to DNA or RNA.For example, the 5′ coding portion of the polynucleotide sequence, whichencodes for the mature polypeptides of the present invention, is used todesign an antisense RNA oligonucleotide of from about 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription (triple-helix, see Leeet al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456(1988); and Dervan et al., Science, 251: 1360 (1991)), therebypreventing transcription and the production of the human chemotacticcytokine. The antisense RNA oligonucleotide hybridizes to the mRNA invivo and blocks translation of the mRNA molecule into the polypeptides(antisense—Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988)). The oligonucleotides described above can also be delivered tocells such that the antisense RNA or DNA may be expressed in vivo toinhibit production of the human chemotactic cytokines.

Another potential human chemotactic cytokine I antagonist is a peptidederivative of the polypeptides which are naturally or syntheticallymodified analogs of the polypeptides that have lost biological functionyet still recognize and bind to the receptors of the polypeptides tothereby effectively block the receptors. Examples of peptide derivativesinclude, but are not limited to, small peptides or peptide-likemolecules.

The antagonists may be employed to inhibit the chemotaxis and activationof macrophages and their precursors, and of neutrophils, basophils, Blymphocytes and some T cell subsets, e.g., activated and CD8 cytotoxic Tcells and natural killer cells, in certain auto-immune and chronicinflammatory and infective diseases. Examples of auto-immune diseasesinclude multiple sclerosis, and insulin-dependent diabetes.

The antagonists may also be employed to treat infectious diseasesincluding silicosis, sarcoidosis, idiopathic pulmonary fibrosis bypreventing the recruitment and activation of mononuclear phagocytes.They may also be employed to treat idiopathic hyper-eosinophilicsyndrome by preventing eosinophil production and migration. Endotoxicshock may also be treated by the antagonists by preventing the migrationof macrophages and their production of the human chemotactic cytokinesof the present invention.

The antagonists may also be employed for treating atherosclerosis, bypreventing monocyte infiltration in the artery wall.

The antagonists may also be employed to treat histamine-mediatedallergic reactions and immunological disorders including late phaseallergic reactions, chronic urticaria, and atopic dermatitis byinhibiting chemokine-induced mast cell and basophil degranulation andrelease of histamine. IgE-mediated allergic reactions such as allergicasthma, rhinitis, and eczema may also be treated.

The antagonists may also be employed to treat chronic and acuteinflammation by preventing the attraction of monocytes to a wound area.They may also be employed to regulate normal pulmonary macrophagepopulations, since chronic and acute inflammatory pulmonary diseases areassociated with sequestration of mononuclear phagocytes in the lung. Inaddition, the antagonists may be employed to treat inflammatory lesions,e.g., rheumatoid arthritis, sarcoid and catscratch granulomas, anddermathopathic lymphadenopathy.

Antagonists may also be employed to treat rheumatoid arthritis bypreventing the attraction of monocytes into synovial fluid in the jointsof patients. Monocyte influx and activation plays a significant role inthe pathogenesis of both degenerative and inflammatory arthropathies.

The antagonists may be employed to interfere with the deleteriouscascades attributed primarily to IL-1 and TNF, which prevents thebiosynthesis of other inflammatory cytokines. In this way, theantagonists may be employed to prevent inflammation. The antagonists mayalso be employed to inhibit prostaglandin-independent fever induced bychemokines.

The antagonists may also be employed to treat cases of bone marrowfailure, for example, aplastic anemia and myelodysplastic syndrome.

The antagonists may also be employed to treat asthma and allergy bypreventing eosinophil accumulation in the lung. The antagonists may alsobe employed to treat subepithelial basement membrane fibrosis which is aprominent feature of the asthmatic lung.

Antagonists may also be employed to treat psoriasis since an elevatedlevel of members of the S100 protein family have been found in theepidermis of psoriatic models.

The antagonists may be employed in a composition with a pharmaceuticallyacceptable carrier, e.g., as hereinafter described.

The human chemotactic cytokines and agonists and antagonists may beemployed in combination with a suitable pharmaceutical carrier. Suchcompositions comprise a therapeutically effective amount of thepolypeptide, and a pharmaceutically acceptable carrier or excipient.Such a carrier includes but is not limited to saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Theformulation should suit the mode of administration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptides and agonists and antagonists may be employed in conjunctionwith other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner such as by the topical, intravenous, intraperitoneal,intramuscular, intratumor, subcutaneous, intranasal or intradermalroutes. The pharmaceutical compositions are administered in an amountwhich is effective for treating and/or prophylaxis of the specificindication. In general, the polypeptides will be administered in anamount of at least about 10 μg/kg body weight and in most cases theywill be administered in an amount not in excess of about 8 mg/Kg bodyweight per day. In most cases, the dosage is from about 10 μg/kg toabout 1 mg/kg body weight daily, taking into account the routes ofadministration, symptoms, etc.

The human chemotactic cytokines, and agonists or antagonists which arepolypeptides, may be employed in accordance with the present inventionby expression of such polypeptides in vivo, which is often referred toas “gene therapy.”

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

Retroviruses from which the retroviral plasmid vectors hereinabovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

The vector includes one or more promoters. Suitable promoters which maybe employed include, but are not limited to, the retroviral LTR; theSV40 promoter; and the human cytomegalovirus (CMV) promoter described inMiller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or anyother promoter (e.g., cellular promoters such as eukaryotic cellularpromoters including, but not limited to, the histone, pol III, andβ-actin promoters). Other viral promoters which may be employed include,but are not limited to, adenovirus promoters, thymidine kinase (TK)promoters, and B19 parvovirus promoters. The selection of a suitablepromoter will be apparent to those skilled in the art from the teachingscontained herein.

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orheterologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the α-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, ψ-2,ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs.5-14 (1990), which is incorporated herein by reference in its entirety.The vector may transduce the packaging cells through any means known inthe art. Such means include, but are not limited to, electroporation,the use of liposomes, and CaPO₄ precipitation. In one alternative, theretroviral plasmid vector may be encapsulated into a liposome, orcoupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the polypeptides.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells. Thesequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clones to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60 bases. For a review of this technique, see Verma etal., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press,New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

Antibodies specific to the polypeptide of the present invention may beemployed as a diagnostic to determine elevated levels of the polypeptidein a sample derived from a host by techniques known in the art. Theseelevated levels are indicative of certain disorders which arecharacterized by elevated levels of the protein of the present inventionand members of its family, for example, lichen planus, lupuserythematosus and psoriasis vulgaris, cystic fibrosis and inflammatorylesions, e.g., rheumatoid arthritis, sarcoid and catscratch granulomasand dermathopathic lymphadenopathy.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice may be used to express humanized antibodies to immunogenicpolypeptide products of this invention.

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples certainfrequently occurring methods and/or terms will be described.

“Plasmids” are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980).

“Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units to T4 DNA ligase (“ligase”)per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

Unless otherwise stated, transformation was performed as described inthe method of Graham, F. and Van der Eb, A., Virology, 52:456-457(1973).

EXAMPLE 1 Bacterial Expression and Purification of Chemotactic Cytokine

The DNA sequence encoding for chemotactic cytokine, ATCC™ #97304, wasinitially amplified using PCR oligonucleotide primers corresponding tothe 5′ and 3′ end sequences of the processed chemotactic cytokine Inucleic acid sequence (minus the putative signal peptide sequence).Additional nucleotides corresponding to the chemotactic cytokine I genewere added to the 5′ and 3′ end sequences respectively. The 5′oligonucleotide primer has the sequence 5′CGC GGA TCC ATG ACA AAA CTT G3′ (SEQ ID NO:3) contains a BamHI restriction enzyme site (bold)followed by 13 nucleotides of chemotactic cytokine I coding sequence(underlined). The 3′ sequence 5′ CGC GGA TCC CTA CTC TTT GTG GGT GTG G3′ (SEQ ID NO:4) contains complementary sequences to a BamHI site (bold)and was followed by 16 nucleotides of gene specific sequences precedingthe termination codon. The restriction enzyme sites correspond to therestriction enzyme sites on the bacterial expression vector pQE-9(Qiagen, Inc. Chatsworth, Calif.). pQE-9 encodes antibiotic resistance(Amp^(r)), a bacterial origin of replication (ori), an IPTG-regulatablepromoter operator (P/O), a ribosome binding site (RBS), a 6-His tag andrestriction enzyme sites. pQE-9 was then digested with BamHI. The codingsequence was amplified using the described primers (SEQ ID NO:3 and 4)and then digested with BamHI. The digested sequences were ligated intopQE-9 and were inserted in frame with the sequence encoding for thehistidine tag and the RBS. The ligation mixture was then used totransform the E. coli strain M15/rep 4 (Qiagen, Inc.) by the proceduredescribed in Sambrook, J. et al., Molecular Cloning: A LaboratoryManual, Cold Spring Laboratory Press, (1989). M15/rep4 contains multiplecopies of the plasmid pREP4, which expresses the lacI repressor and alsoconfers kanamycin resistance (Kan^(r)). Transformants were identified bytheir ability to grow on LB plates and ampicillin/kanamycin resistantcolonies were selected. Plasmid DNA was isolated and confirmed byrestriction analysis. Clones containing the desired constructs weregrown overnight (O/N) in liquid culture in LB media supplemented withboth Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture was used toinoculate a large culture at a ratio of 1:100 to 1:250. The cells weregrown to an optical density 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG(“Isopropyl-B-D-thiogalacto pyranoside”) was then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/O leading to increased gene expression. Cells were grownan extra 3 to 4 hours. Cells were then harvested by centrifugation. Thecell pellet was solubilized in the chaotropic agent 6 Molar GuanidineHCl pH 8.0. After clarification, solubilized chemotactic cytokine I waspurified from this solution by chromatography on a Nickel-Chelate column(Hochuli, E. et al., J. Chromatography 411:177-184 (1984)) underconditions that allow for tight binding by proteins containing the 6-Histag. Chemotactic cytokine I (>90% pure) was eluted from the column in 6Mguanidine HCl. Protein renaturation out of GnHCl can be accomplished byseveral protocols (Jaenicke, R. and Rudolph, R., Protein Structure—APractical Approach, IRL Press, New York (1990)). Initially, stepdialysis was utilized to remove the GnHCL. Alternatively, the purifiedprotein isolated from the Ni-chelate column can be bound to a secondcolumn over which a decreasing linear GnHCL gradient was run. Theprotein was allowed to renature while bound to the column and wassubsequently eluted with a buffer containing 250 mM Imidazole, 150 mMNaCl, 25 mM Tris-HCl pH 7.5 and 10% Glycerol. Finally, soluble proteinwas dialyzed against a storage buffer containing 5 mM AmmoniumBicarbonate.

EXAMPLE 2 Expression of Recombinant Chemotactic Cytokine I in COS Cells

The expression of plasmid, chemotactic cytokine I HA is derived from avector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin ofreplication, 2) ampicillin resistance gene, 3) E. coli replicationorigin, 4) CMV promoter followed by a polylinker region, a SV40 intronand polyadenylation site. A DNA fragment encoding the entire chemotacticcytokine I precursor and a HA tag fused in frame to its 3′ end is clonedinto the polylinker region of the vector, therefore, the recombinantprotein expression is directed under the CMV promoter. The HA tagcorrespond to an epitope derived from the influenza hemagglutininprotein as previously described (I. Wilson, H. Niman, R. Heighten, ACherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767). The infusionof HA tag to the target protein allows easy detection of the recombinantprotein with an antibody that recognizes the HA epitope.

The plasmid construction strategy is described as follows:

The DNA sequence encoding for chemotactic cytokine, ATCC™ #97304, isconstructed by PCR using two primers: the 5′ primer 51GCGCGGATCCACCATGACAAAACTTGAAGAG 3′ (SEQ ID NO:5) contains a BamHI site(in bold) followed by 15 nucleotides of chemotactic cytokine I codingsequence starting from the minus 3 position relative to initiationcodon; the 3′ sequence 5′GCGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTACTCTTTG TGGGTGTG 3′ (SEQ IDNO:6) contains complementary sequences to an XbaI site, translation stopcodon, HA tag and the last 15 nucleotides of the chemotactic cytokine Icoding sequence (not including the stop codon). Therefore, the PCRproduct contains a BamHI site, chemotactic cytokine I coding sequencefollowed by HA tag fused in frame, a translation termination stop codonnext to the HA tag, and an XbaI site. The PCR amplified DNA fragment andthe vector, pcDNAI/Amp, are digested with BamHI and XbaI restrictionenzyme and ligated. The ligation mixture is transformed into E. colistrain SURE (Stratagene Cloning Systems, La Jolla, Calif.) thetransformed culture is plated on ampicillin media plates and resistantcolonies are selected. Plasmid DNA is isolated from transformants andexamined by restriction analysis for the presence of the correctfragment. For expression of the recombinant chemotactic cytokine, COScells are transfected with the expression vector by DEAE-DEXTRAN method(J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A LaboratoryManual, Cold Spring Laboratory Press, (1989)). The expression of thechemotactic cytokine I HA protein is detected by radiolabelling andimmunoprecipitation method (E. Harlow, D. Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, (1988)). Cells are labeledfor 8 hours with ³⁵S-cysteine two days post transfection. Culture mediaare then collected and cells are lysed with detergent (RIPA buffer (150mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5).(Wilson, I. et al., Id. 37:767 (1984)). Both cell lysate and culturemedia are precipitated with a HA specific monoclonal antibody. Proteinsprecipitated are analyzed by SDS-PAGE.

EXAMPLE 3 Cloning and Expression of Chemotactic Cytokine I Using theBaculovirus Expression System

The DNA sequence encoding the full length chemotactic cytokine Iprotein, ATCC™ #97304, is amplified using PCR oligonucleotide primerscorresponding to the 5′ and 3′ sequences of the gene:

The 5′ primer has the sequence 5′ CGC GGA TCC CAC AAA ACT TGA AGA GCA TC3′ (SEQ ID NO:7) and contains a BamHI restriction enzyme site (in bold)followed by 19 nucleotides of coding sequence.

The 3′ primer has the sequence 5′ CGC GGA TCC CTA CTC TTT GTG GGT GTG G3′ (SEQ ID NO:8) and contains the cleavage site for the restrictionendonuclease BamHI and 16 nucleotides complementary to the 3′ translatedsequence of the chemotactic cytokine I gene and stop codon. Theamplified sequences are isolated from a 1% agarose gel using acommercially available kit (“Geneclean™,” BIO 101 Inc., La Jolla,Calif.). The fragment is then digested with the endonucleases BamHI,then purified again on a 1% agarose gel. This fragment is designated F2.

The vector pA2-GP (modification of pVL941 vector, discussed below) isused for the expression of the chemotactic cytokine I protein using thebaculovirus expression system (for review see: Summers, M. D. and Smith,G. E. 1987, A manual of methods for baculovirus vectors and insect cellculture procedures, Texas Agricultural Experimental Station Bulletin No.1555). This expression vector contains the strong polyhedrin promoter ofthe Autographa californica nuclear polyhedrosis virus (AcMNPV) followedby the recognition sites for the restriction endonucleases BamHI andAsp781. The polyadenylation site of the simian virus (SV)40 is used forefficient polyadenylation. For an easy selection of recombinant virusesthe beta-galactosidase gene from E. coli is inserted in the sameorientation as the polyhedrin promoter followed by the polyadenylationsignal of the polyhedrin gene. The polyhedrin sequences are flanked atboth sides by viral sequences for the cell-mediated homologousrecombination of cotransfected wild-type viral DNA. Many otherbaculovirus vectors could be used in place of pA2-GP such as pAc373,pVL941 if a signal peptide is added to the coding sequence as described(Luckow, V. A. and Summers, M. D., Virology, 170:31-39).

The plasmid is digested with the restriction enzymes BamHI and thendephosphorylated using calf intestinal phosphatase by procedures knownin the art. The DNA is then isolated from a 1% agarose gel using thecommercially available kit (“Geneclean™” BIO 101 Inc., La Jolla,Calif.). This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNAligase. E. coli HB101 cells are then transformed and bacteria identifiedthat contained the plasmid (pBac-chemotactic cytokine) with thechemotactic cytokine I gene using the enzymes BamHI and Asp781. Thesequence of the cloned fragment is confirmed by DNA sequencing.

5 μg of the plasmid pBac-chemotactic cytokine I is cotransfected with1.0 μg of a commercially available linearized baculovirus (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.) using the lipofectionmethod (Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBac-chemotacticcytokine I are mixed in a sterile well of a microtiter plate containing50 μl of serum free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards 10 μl Lipofectin plus 90 μl Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added dropwise to the Sf9(Spodoptera frugiperda) insect cells (ATCC™ CRL 1711) seeded in a 35 mmtissue culture plate with 1 ml Grace's medium without serum. The plateis rocked back and forth to mix the newly added solution. The plate isthen incubated for 5 hours at 27° C. After 5 hours the transfectionsolution is removed from the plate and 1 ml of Grace's insect mediumsupplemented with 10% fetal calf serum is added. The plate is put backinto an incubator and cultivation continued at 27° C. for four days.

After four days the supernatant is collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) is used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

Four days after the serial dilution, the viruses are added to the cellsand blue stained plaques are picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses is then resuspendedin an Eppendorf tube containing 200 μl of Grace's medium. The agar isremoved by a brief centrifugation and the supernatant containing therecombinant baculovirus is used to infect Sf9 cells seeded in 35 mmdishes. Four days later the supernatants of these culture dishes areharvested and then stored at 4° C.

Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-chemotactic cytokine I at a multiplicity of infection(MOI) of 2. Six hours later the medium is removed and replaced withSF900 II medium minus methionine and cysteine (Life Technologies Inc.,Gaithersburg). 42 hours later 5 μCi of ³⁵S-methionine and 5 μCi ³⁵Scysteine (Amersham) are added. The cells are further incubated for 16hours.

The growth medium was harvested on day 4 post-infection. After theremoval of baculovirus cells by continuous centrifugation, thesupernatant was applied to a cation exchange column (pros HS 50 resinfrom PerSeptive Biosystem) pre-equilibrated with buffer A (40 mM SodiumAcetate, 50 mM NaCl, pH 5.5). The column was eluted stepwise withincreasing NaCl concentration in the same buffer. The fractionscontaining the human chemotactic cytokine I were pooled, diluted withbuffer A and applied to another cation exchange column (CM20), followedby a NaCl gradient elution. The combined fractions were further purifiedby gel filtration chromatography (Superdex 200 or Superdex 75 fromPharmacia Biotech). Human chemotactic cytokine I was finally purified tohomogeneity (95%) and concentrated by cation exchange chromatography(CM20) after the gel filtration fractionation.

EXAMPLE 4 Expression Via Gene Therapy

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention is amplifiedusing PCR primers which correspond to the 5′ and 3′ end sequencesrespectively. The 5′ primer containing an EcoRI site and the 3′ primerfurther includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is used to transformbacteria HB101, which are then plated onto agar-containing kanamycin forthe purpose of confirming that the vector had the gene of interestproperly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellsare transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

1. An isolated polypeptide consisting of at least 30 contiguous aminoacid residues of SEQ ID NO:2.
 2. The isolated polypeptide of claim 1,wherein said polypeptide consists of at least 50 contiguous amino acidresidues of SEQ ID NO:2.
 3. An isolated polypeptide comprising aminoacids 2 to 92 of SEQ ID NO:2.
 4. The isolated polypeptide of claim 3,wherein said polypeptide comprises amino acids 1 to 92 of SEQ ID NO:2.5. The isolated polypeptide of claim 1 fused to a heterologouspolypeptide.
 6. An isolated polypeptide consisting of at least 30contiguous amino acid residues of the polypeptide encoded by the humancDNA contained in ATCC™ Deposit No.
 97304. 7. The isolated polypeptideof claim 6, wherein said polypeptide consists of at least 50 contiguousamino acid residues of the polypeptide encoded by the human cDNAcontained in ATCC™ Deposit No.
 97304. 8. An isolated polypeptidecomprising the mature polypeptide encoded by the human cDNA contained inATCC™ Deposit No.
 97304. 9. An isolated polypeptide comprising the fulllength polypeptide encoded by the human cDNA contained in ATCC™ DepositNo.
 97304. 10. The isolated polypeptide of claim 6 fused to aheterologous polypeptide.
 11. An isolated protein produced by the methodcomprising: (a) culturing a host cell under conditions suitable toproduce the protein molecule of claim 1, and (b) recovering said proteinmolecule.
 12. An isolated protein produced by the method comprising: (a)culturing a host cell under conditions suitable to produce the proteinmolecule of claim 2; and (b) recovering said protein molecule.
 13. Anisolated protein produced by the method comprising: (a) culturing a hostcell under conditions suitable to produce the protein molecule of claim3; and (b) recovering said protein molecule.
 14. An isolated proteinproduced by the method comprising: (a) culturing a host cell underconditions suitable to produce the protein molecule of claim 4; and (b)recovering said protein molecule.
 15. An isolated protein produced bythe method comprising: (a) culturing a host cell under conditionssuitable to produce the protein molecule of claim 5, and (b) recoveringsaid protein molecule.
 16. An isolated protein produced by the methodcomprising: (a) culturing a host cell under conditions suitable toproduce the protein molecule of claim 6; and (b) recovering said proteinmolecule.
 17. An isolated protein produced by the method comprising: (a)culturing a host cell under conditions suitable to produce the proteinmolecule of claim 7; and (b) recovering said protein molecule.
 18. Anisolated protein produced by the method comprising: (a) culturing a hostcell under conditions suitable to produce the protein molecule of claim8; and (b) recovering said protein molecule.
 19. An isolated proteinproduced by the method comprising: (a) culturing a host cell underconditions suitable to produce the protein molecule of claim 9, and (b)recovering said protein molecule.
 20. An isolated protein produced bythe method comprising: (a) culturing a host cell under conditionssuitable to produce the protein molecule of claim 10, and (b) recoveringsaid protein molecule.